Effect of Sodium Selenate and Selenocystine on Savoy Cabbage Yield, Morphological and Biochemical Characteristics under Chlorella Supply

Biofortification of Brassica oleracea with selenium (Se) is highly valuable both for human Se status optimization and functional food production with direct anti-carcinogenic activity. To assess the effects of organic and inorganic Se supply for biofortifying Brassica representatives, foliar applications of sodium selenate and selenocystine (SeCys2) were performed on Savoy cabbage treated with the growth stimulator microalgae Chlorella. Compared to sodium selenate, SeCys2 exerted a stronger growth stimulation of heads (1.3 against 1.14 times) and an increase of leaf concentration of chlorophyll (1.56 against 1.2 times) and ascorbic acid (1.37 against 1.27 times). Head density was reduced by 1.22 times by foliar application of sodium selenate and by 1.58 times by SeCys2. Despite the greater growth stimulation effect of SeCys2, its application resulted in lower biofortification levels (2.9 times) compared to sodium selenate (11.6 times). Se concentration decreased according to the following sequence: leaves > roots > head. The antioxidant activity (AOA) was higher in water extracts compared to the ethanol ones in the heads, but the opposite trend was recorded in the leaves. Chlorella supply significantly increased the efficiency of biofortification with sodium selenate (by 1.57 times) but had no effect in the case of SeCys2 application. Positive correlations were found between leaf and head weight (r = 0.621); head weight and Se content under selenate supply (r = 0.897–0.954); leaf ascorbic acid and total yield (r = 0.559), and chlorophyll (r = +0.83–0.89). Significant varietal differences were recorded for all the parameters examined. The broad comparison performed between the effects of selenate and SeCys2 showed significant genetic differences as well as important peculiarities connected with the Se chemical form and its complex interaction with Chlorella treatment.


Introduction
The biofortification of agricultural crops with selenium (Se) is one of the most interesting methods to optimize human Se status [1]. Many years of experience regarding Se-enriched mineral fertilizers in Finland were accompanied by a significant decrease in population mortality due to cardiovascular diseases and cancer [2]. Indeed, this approach entails the production of functional food with high Se levels and enhanced the concentration of other natural antioxidants, valuable for health maintenance [3]. In biological systems, Se easily substitutes sulfur in organic compounds, indicating high biofortification prospects Though Chlorella is known to improve plant nutrition and alleviate environmental stress [28], no beneficial effect of Chlorella application was recorded in the present work. Chlorella supplementation did not affect the mean head weight and marketable yield, but the increase of mean head weight and marketable yield as a result of joint Se + Chlorella supply compared to control plants exclusively reflected the beneficial effect of organic and inorganic Se forms (Table 1). Furthermore, all the treatments applied, i.e., Se and/or Chlorella, significantly increased the Savoy cabbage leaf weight, to a remarkable extent under joint selenate + Chlorella application.
The growth-promoting effect of Se biofortification using low concentrations of Se has been previously recorded in many plant species [30,31], though cabbage biofortification with SeCys 2 has been performed in the present research for the first time.
Taking into account the equation describing oblate spheroid volume and the head weight data related to the Savoy cabbage, we assessed the effects of single and joint application of different Se forms and microalgae Chlorella on the Savoy cabbage head density ( Figure 1).  The data presented in Figure 1 reveal the phenomenon of a significant head den decrease as a result of Chlorella, inorganic and especially organic Se treatments, with values decreasing under SeCys2 supply depending on the cultivar: by 1.52 (Vertu 134 1.59 (Melissa) and 1.64 times (Golubtsy).
Interestingly, the joint sodium selenate and Chlorella supply did not significantl fect the head density compared to Se treatment, whereas the joint application of Se and Chlorella significantly enhanced the head density. It may be supposed that bot forms delay the Savoy cabbage senescence, as was previously demonstrated in other p species [30], via regulation of antioxidant enzymes' activity, downregulation of ethy production [32], and increasing chlorophyll content [33].
The results indicate the contrasting trends between head density and market yield (Table 1, Figure 1). In this respect, it may be inferred that though SeCys2 treatm significantly improved yield, at the same time it decreased the head density. In all c vars, the density of control plant heads was either greater (cv. Melissa) or tendent higher compared to the other treatments. Positive correlation coefficients were recor between leaves and head weight (r = 0.621, p < 0.05); total and leaves weight (r = 0.625 0.05) and total and head weight (r = 0.984, p < 0.001). In contrast, negative correlat were found between head density, total weight, and mean head weight (r = 0.571 to 0 p < 0.05).

Selenium Accumulation
The data of the present investigation indicate that Savoy cabbage Se biofortifica provided significant accumulation of this microelement, with concentrations depen on Se chemical form and Chlorella supply ( Figure 2). Previous investigations demonstr that Brassica oleracea representatives are able to convert sodium selenate to selenome nine [34]. Selenocystine is reportedly assimilated by plants via amino acid transporter contrast to inorganic selenate, which is absorbed through the sulfate pathway [35]. The data presented in Figure 1 reveal the phenomenon of a significant head density decrease as a result of Chlorella, inorganic and especially organic Se treatments, with the values decreasing under SeCys 2 supply depending on the cultivar: by 1.52 (Vertu 1340) to 1.59 (Melissa) and 1.64 times (Golubtsy).
Interestingly, the joint sodium selenate and Chlorella supply did not significantly affect the head density compared to Se treatment, whereas the joint application of SeCys 2 and Chlorella significantly enhanced the head density. It may be supposed that both Se forms delay the Savoy cabbage senescence, as was previously demonstrated in other plant species [30], via regulation of antioxidant enzymes' activity, downregulation of ethylene production [32], and increasing chlorophyll content [33].
The results indicate the contrasting trends between head density and marketable yield (Table 1, Figure 1). In this respect, it may be inferred that though SeCys 2 treatment significantly improved yield, at the same time it decreased the head density. In all cultivars, the density of control plant heads was either greater (cv. Melissa) or tendentially higher compared to the other treatments. Positive correlation coefficients were recorded between leaves and head weight (r = 0.621, p < 0.05); total and leaves weight (r = 0.625, p < 0.05) and total and head weight (r = 0.984, p < 0.001). In contrast, negative correlations were found between head density, total weight, and mean head weight (r = 0.571 to 0.580, p < 0.05).

Selenium Accumulation
The data of the present investigation indicate that Savoy cabbage Se biofortification provided significant accumulation of this microelement, with concentrations depending on Se chemical form and Chlorella supply ( Figure 2). Previous investigations demonstrated that Brassica oleracea representatives are able to convert sodium selenate to selenomethionine [34]. Selenocystine is reportedly assimilated by plants via amino acid transporters, in contrast to inorganic selenate, which is absorbed through the sulfate pathway [35].  As can be seen in Figures 2 and 3, Chlorella supply showed complex effects. Indeed, it did not affect Se accumulation in control plants but significantly improved Se levels in case of joint selenate + Chlorella application. Selenocystine treatment under Chlorella supply elicited varietal differences: significant increase of Se level in Vertu 1340 roots, and in Melissa roots, head, and leaves. However, only in cv. Melissa, Chlorella demonstrated significant Se increase in all plant parts upon the joint applications of selenate + Chlorella and SeCys 2 + Chlorella ( Figure 2). As can be seen in Figures 2 and 3, Chlorella supply showed complex effects. Ind it did not affect Se accumulation in control plants but significantly improved Se leve case of joint selenate + Chlorella application. Selenocystine treatment under Chlorella ply elicited varietal differences: significant increase of Se level in Vertu 1340 roots, a Melissa roots, head, and leaves. However, only in cv. Melissa, Chlorella demonstrated nificant Se increase in all plant parts upon the joint applications of selenate + Chlorella SeCys2 + Chlorella ( Figure 2).  The analysis of biofortification levels revealed the predominance of leaf Se accumulation, especially in case of joint selenate + Chlorella supply ( Figure 3A-C). Much less significant effect or even its absence was recorded for roots and especially head biofortification levels.
The presented data suggest that the foliar Se supply of Savoy cabbage resulted in predominant Se accumulation in leaves, with the highest concentration in sodium selenate treated plants, while SeCys2 led to much lower levels of Se biofortification. Figures 2 and  3 show that Chlorella application may significantly increase organic and inorganic Se accumulation in leaves, head, and roots only in cultivar Melissa, with the predominance of leaf and root Se increase in cv. Vertu 1340, both under sodium selenate and SeCys2 supply, whereas in cv. Golubtsy, Chlorella supply was beneficial only to improve sodium selenate assimilation.
Notably, microalgae reportedly improve plant nutrition [28] but their effect on Se accumulation has never been investigated. Chlorella ability to increase sodium selenate biofortification levels is of great importance to produce functional food with high content of this element. On the other hand, the complexity of Chlorella-SeCys2 interaction demonstrates the need for further investigations.
Taking into account the beneficial effect of Chlorella supply only on selenate accumulation in Savoy cabbage, a relationship between head Se levels and weight has been evaluated based on the results obtained in 2021 and 2022 ( Figure 4). The analysis of biofortification levels revealed the predominance of leaf Se accumulation, especially in case of joint selenate + Chlorella supply ( Figure 3A-C). Much less significant effect or even its absence was recorded for roots and especially head biofortification levels.
The presented data suggest that the foliar Se supply of Savoy cabbage resulted in predominant Se accumulation in leaves, with the highest concentration in sodium selenate treated plants, while SeCys 2 led to much lower levels of Se biofortification. Figures 2 and 3 show that Chlorella application may significantly increase organic and inorganic Se accumulation in leaves, head, and roots only in cultivar Melissa, with the predominance of leaf and root Se increase in cv. Vertu 1340, both under sodium selenate and SeCys 2 supply, whereas in cv. Golubtsy, Chlorella supply was beneficial only to improve sodium selenate assimilation.
Notably, microalgae reportedly improve plant nutrition [28] but their effect on Se accumulation has never been investigated. Chlorella ability to increase sodium selenate biofortification levels is of great importance to produce functional food with high content of this element. On the other hand, the complexity of Chlorella-SeCys 2 interaction demonstrates the need for further investigations.
Taking into account the beneficial effect of Chlorella supply only on selenate accumulation in Savoy cabbage, a relationship between head Se levels and weight has been evaluated based on the results obtained in 2021 and 2022 ( Figure 4).  The analysis of biofortification levels revealed the predominance of leaf Se accumulation, especially in case of joint selenate + Chlorella supply ( Figure 3A-C). Much less significant effect or even its absence was recorded for roots and especially head biofortification levels.
The presented data suggest that the foliar Se supply of Savoy cabbage resulted in predominant Se accumulation in leaves, with the highest concentration in sodium selenate treated plants, while SeCys2 led to much lower levels of Se biofortification. Figures 2 and  3 show that Chlorella application may significantly increase organic and inorganic Se accumulation in leaves, head, and roots only in cultivar Melissa, with the predominance of leaf and root Se increase in cv. Vertu 1340, both under sodium selenate and SeCys2 supply, whereas in cv. Golubtsy, Chlorella supply was beneficial only to improve sodium selenate assimilation.
Notably, microalgae reportedly improve plant nutrition [28] but their effect on Se accumulation has never been investigated. Chlorella ability to increase sodium selenate biofortification levels is of great importance to produce functional food with high content of this element. On the other hand, the complexity of Chlorella-SeCys2 interaction demonstrates the need for further investigations.
Taking into account the beneficial effect of Chlorella supply only on selenate accumulation in Savoy cabbage, a relationship between head Se levels and weight has been evaluated based on the results obtained in 2021 and 2022 ( Figure 4).  Though the SeCys2 treatment improved head weight more significantly than sodium selenate (Table 1), the determination of the appropriate correlation coefficients is exigent due to (i) lack of Chlorella beneficial effect on organic Se accumulation and (ii) significant varietal differences in plant response to Se/Chlorella supply, which excludes the possibility of calculating a single correlation coefficient for all three cultivars.
More remarkable beneficial effect of SeCys2 on the Savoy cabbage growth and development, compared to sodium selenate, is connected with the well-known growth stimulation effect of amino acids [36,37]. Among amino acids, Cys acts either as a chelating agent accelerating the absorption and transport of microelements within plants, or as an important component of plant enzymes. In this respect, it may be supposed that the highest efficiency of Cys/SeCys2 supply may be obtained in sulfur-accumulator plants, such as Brassica and Allium species. Indeed, recent investigations indicate that Cys significantly stimulates biologically active compounds synthesis in garlic, improving allicin content [38].

Antioxidants
The response of plant antioxidants to Se and Chlorella treatments was rather specific. Though the SeCys 2 treatment improved head weight more significantly than sodium selenate (Table 1), the determination of the appropriate correlation coefficients is exigent due to (i) lack of Chlorella beneficial effect on organic Se accumulation and (ii) significant varietal differences in plant response to Se/Chlorella supply, which excludes the possibility of calculating a single correlation coefficient for all three cultivars.
More remarkable beneficial effect of SeCys 2 on the Savoy cabbage growth and development, compared to sodium selenate, is connected with the well-known growth stimulation effect of amino acids [36,37]. Among amino acids, Cys acts either as a chelating agent accelerating the absorption and transport of microelements within plants, or as an important component of plant enzymes. In this respect, it may be supposed that the highest efficiency of Cys/SeCys 2 supply may be obtained in sulfur-accumulator plants, such as Brassica and Allium species. Indeed, recent investigations indicate that Cys significantly stimulates biologically active compounds synthesis in garlic, improving allicin content [38].

Antioxidants
The response of plant antioxidants to Se and Chlorella treatments was rather specific.

Photosynthetic Pigments
At low concentrations, Se is known to stimulate the biosynthesis of photosynthetic pigments [30], which is directly connected with its growth promoting effect. The data presented in Table 2 indicate significant increase in chlorophyll and carotene content due to Se treatment either singly or in combination with Chlorella supply. It is important that SeCys 2 resulted in a more remarkable increase of photosynthetic pigments accumulation compared to sodium selenate, both singly and in combination with Chlorella. In this respect, the chlorophyll a/b ratio was the highest in plants treated with SeCys 2 , reaching 1. Chlorella vulgaris is one of the most commercially available microalgae. Its utilization in agriculture is connected with its ability to provide simultaneously biofertilization, biostimulation and protection of plants against biotic and abiotic stresses [28]. Chlorella is known to improve cell metabolism, nucleic acid synthesis, photosynthesis, antioxidant status of plants, and water availability [39]. The results of the present work show that the beneficial effect of Chlorella supply is variety dependent. Indeed, the microalgae improved significantly chlorophyll 'a' and carotene content in Melissa leaves, only chlorophyll 'a' content in Vertu 1340, and had no effect on photosynthetic pigments accumulation in Golubtsy. In the present study, the lack of significant Chlorella effect on photosynthetic pigments accumulation and head weight may be connected with the low concentration applied, as 10-fold higher concentrations were shown to increase significantly yield and chlorophyll content in Swiss chard [40]. At the same time, it should be highlighted that significant biomass increase as a result of Chlorella supply was recorded only for the outer leaves of Savoy cabbage (Table 1).
A greater beneficial effect on photosynthetic pigments accumulation was recorded under Se application both singly or in combination with Chlorella ( Figure 5). The data shown in Figure 5 indicate higher beneficial effect of SeCys 2 on total chlorophyll content compared to inorganic form of Se, with the highest expression in Vertu 1340 leaves and the lowest in Golubtsy. Golubtsy. In the present study, the lack of significant Chlorella effect on photosynthetic pigments accumulation and head weight may be connected with the low concentration applied, as 10-fold higher concentrations were shown to increase significantly yield and chlorophyll content in Swiss chard [40]. At the same time, it should be highlighted that significant biomass increase as a result of Chlorella supply was recorded only for the outer leaves of Savoy cabbage (Table 1). A greater beneficial effect on photosynthetic pigments accumulation was recorded under Se application both singly or in combination with Chlorella ( Figure 5). The data shown in Figure 5 indicate higher beneficial effect of SeCys2 on total chlorophyll content compared to inorganic form of Se, with the highest expression in Vertu 1340 leaves and the lowest in Golubtsy. Furthermore, organic Se demonstrated a stronger beneficial effect on carotenoids accumulation in Savoy cabbage leaves ( Table 2). Carotenoids have a major function to protect chlorophyll and the surrounding cells from light damage converting the excess excitation energy to heat [41]. The beneficial effect of Se on photosynthetic pigments accumulation is in accordance with the known increase of the photosystem II quantum efficiency in plants due to Se supply [42][43][44]. The increase of Savoy cabbage biomass under Se biofortification agrees with the mentioned results.
The comparison of variation coefficients (CV) for the morphological parameters tested (Table 1) and photosynthetic pigments accumulation (Table 2) indicated that, among the three cultivars examined, Vertu 1340 was the most sensitive to Se/Chlorella treatment effect on leaf weight and chlorophyll accumulation, the CV values reaching 20.7% and 25% respectively, while the cultivars Golubtsy and Melissa demonstrated much lower variations of the parameters tested.

Ascorbic Acid
Improvement of the ascorbic acid biosynthesis due to Se application is directly connected with the intensity of photosynthesis. The data presented in Table 3 indicate the highest beneficial effect of Se supply on the ascorbic acid accumulation by cvs. Vertu 1340 and Golubtsy and no statistically significant changes of this parameter in cv. Melissa. The Furthermore, organic Se demonstrated a stronger beneficial effect on carotenoids accumulation in Savoy cabbage leaves ( Table 2). Carotenoids have a major function to protect chlorophyll and the surrounding cells from light damage converting the excess excitation energy to heat [41]. The beneficial effect of Se on photosynthetic pigments accumulation is in accordance with the known increase of the photosystem II quantum efficiency in plants due to Se supply [42][43][44]. The increase of Savoy cabbage biomass under Se biofortification agrees with the mentioned results.
The comparison of variation coefficients (CV) for the morphological parameters tested (Table 1) and photosynthetic pigments accumulation (Table 2) indicated that, among the three cultivars examined, Vertu 1340 was the most sensitive to Se/Chlorella treatment effect on leaf weight and chlorophyll accumulation, the CV values reaching 20.7% and 25% respectively, while the cultivars Golubtsy and Melissa demonstrated much lower variations of the parameters tested.

Ascorbic Acid
Improvement of the ascorbic acid biosynthesis due to Se application is directly connected with the intensity of photosynthesis. The data presented in Table 3 indicate the highest beneficial effect of Se supply on the ascorbic acid accumulation by cvs. Vertu 1340 and Golubtsy and no statistically significant changes of this parameter in cv. Melissa. The ascorbic acid levels in Golubtsy leaves increased under Se supply by 1.31-1.60 times and in heads by 1.25-1.48 times. The corresponding changes were recorded in Vertu 1340 leaves treated with sodium selenate, SeCys 2 , and a combination of SeCys 2 + Chlorella (1.41-1.50 time). Significant changes of ascorbic acid content in Vertu 1340 were demonstrated only for plants treated with SeCys 2 (1.28 times). In general, the present results indicate the low effect of Chlorella foliar supply on the ascorbic acid biosynthesis in Savoy cabbage. Ascorbic acid is an important cofactor for several enzymes involved in the synthesis of numerous secondary metabolites, including phytohormones, and a component in plant protection against environmental stresses [45]. In the present investigation, the highest content of ascorbic acid in leaves and head of Savoy cabbage corresponded to the highest leaves and head biomass ( Table 1). The ascorbic acid increase due to Se supplementation was previously recorded in several leafy green vegetables [46][47][48], and enhanced plant Fe and Zn bioavailability [49]. The present data indicate that SeCys 2 /Chlorella supply increased the ascorbic acid content by 18-44% in Savoy cabbage head and by 21-60% in leaves, compared to control plants. Furthermore, the ascorbic acid biosynthesis in the Savoy cabbage leaves was positively correlated with the intensity of photosynthetic pigments accumulation ( Figure 6). A similar relationship between ascorbic acid and photosynthetic pigments biosynthesis was previously found in canola exposed to salt stress [50]. Ascorbic acid demonstrates a significant role in the photosynthesis maintenance and in the protection of the photosynthetic apparatus against reactive oxygen species and photoinhibition [51]. According to the present results, the coefficient of correlation between the ascorbic acid content in leaves and the total Savoy cabbage biomass was 0.559 (p < 0.03).

Total Antioxidant Activity (AOA)
Many plants contain more hydrophobic antioxidants than hydrophilic ones, which stimulates the utilization of 70% ethanol extraction during antioxidant activity determination [52].
The present results indicate significant peculiarities in hydrophilic and hydrophobic antioxidant distribution in the leaves and head of the Savoy cabbage. Table 4 indicates the predominance of ethanol soluble antioxidants in leaves, and water soluble in the head. Moreover, significant differences between the levels of water and ethanol soluble antioxidants in leaves/ head regard predominantly the plants treated with Se and Chlorella, but not control plants. Significant accumulation levels of hydrophilic antioxidants in Brassica oleracea representatives were previously recorded by Podsedek et al. [53] in cabbage heads, but no information was given about the leaves and roots antioxidants distribution. The results of the present investigation demonstrated significant differences in hydrophobic and hydrophilic antioxidants content only in roots of control plants (Table 4, Figure 7). It may be supposed that Se/Chlorella treatments stimulate the mobility of root hydrophilic antioxidants and their transport to heads. According to the present results, the coefficient of correlation between the ascorbic acid content in leaves and the total Savoy cabbage biomass was 0.559 (p < 0.03).

Total Antioxidant Activity (AOA)
Many plants contain more hydrophobic antioxidants than hydrophilic ones, which stimulates the utilization of 70% ethanol extraction during antioxidant activity determination [52].
The present results indicate significant peculiarities in hydrophilic and hydrophobic antioxidant distribution in the leaves and head of the Savoy cabbage. Table 4 indicates the predominance of ethanol soluble antioxidants in leaves, and water soluble in the head. Moreover, significant differences between the levels of water and ethanol soluble antioxidants in leaves/ head regard predominantly the plants treated with Se and Chlorella, but not control plants. Significant accumulation levels of hydrophilic antioxidants in Brassica oleracea representatives were previously recorded by Podsedek et al. [53] in cabbage heads, but no information was given about the leaves and roots antioxidants distribution. The results of the present investigation demonstrated significant differences in hydrophobic and hydrophilic antioxidants content only in roots of control plants (Table 4, Figure 7). It may be supposed that Se/Chlorella treatments stimulate the mobility of root hydrophilic antioxidants and their transport to heads.    The fact that plant phenolics content (Table 5) did not differ significantly between the different experimental treatments suggests that AOA differences between Savoy cabbage leaves and head are not connected directly with the polyphenol profile of plants. The main phenolic acids in Savoy cabbage are gallic, sinapic, and chlorogenic, while quercetin and kaempferol were detected among flavonoids [54]. Heimler et al. [55] reported that, among different Brassica oleracea species, Savoy cabbage showed one of the highest levels of apigenin and luteolin [55]. Notably, the differences in antioxidant activity between water and 70% ethanol extracts are not connected either with polyphenols accumulation or ascorbic acid content due to the full degradation of the vitamin at 80 • C extraction for 1 h. In this respect, it may be supposed that water-soluble glucosinolates of the Savoy cabbage prevail in cabbage head compared to leaves. However, further investigations are needed to prove the above hypothesis. In 1979, VanEtten showed the predominance of oxidized glucosinolates forms in the Savoy cabbage head [56].

Dry Matter, Nitrates, Total Dissolved Solids (TDS), Carbohydrates
No significant differences were detected between the different experimental treatments in terms of dry matter, nitrates, total dissolved solids, and carbohydrates content ( Table 6). The leaves/head dry matter ratio changed from 1.61 to 1.74 and 1.74 (cultivars Vertu 1340, Golubtsy, and Melissa, respectively). The corresponding nitrate leaves/head ratio differed from 0.95 to 0.75 and to 0.80. The highest leaf dry matter and nitrate content were recorded in cultivar Golubtsy. The total dissolved solids (TDS) reflect the content of water-soluble carbohydrates, minerals, nitrates, amino acids, and proteins. The high stability of TDS in cabbage leaves and heads in the present study is in accordance with the stability of nitrates and carbohydrates, both mono-and disaccharides (Table 6). In this respect, among the cultivars tested, Vertu 1340 was characterized by the highest sugar levels.
The active participation of Se in nitrogen metabolism via stimulation of the amino acid biosynthesis and increase of the nitrate reductase and glutamate synthase activity [46,57,58] did not arise in Savoy cabbage upon Se biofortification. Notably, Se-nitrogen interaction is species-specific and is significantly affected by plant hormonal status. In this respect, the lack of significant changes in the nitrate levels as a result of sodium selenate supplementation was recorded previously in kohlrabi [15] while intensive nitrate content decrease was found in wheat [59], sunflower [60], Indian mustard [46], lettuce [58,61,62], and potatoes [63]. The possibility of the hormonal factor participation in Se-nitrate reductase interaction was previously revealed in male and female forms of spinach plants [64]. It may be supposed that higher concentrations of sodium selenate and SeCys 2 may cause significant changes in nitrates accumulation in Savoy cabbage, but this topic needs further investigation.
The values of mean temperature and relative humidity during the crop cycles are presented in Table 7. The sowing was performed on 27-29 April in multicell containers and the seedling planting on 2-4 June with a density of 2.9 plants per m -2 (50 × 70 cm).
The experimental treatments, applied to three Savoy cabbage cultivars (Vertu 1340, Golubtsy, Melissa), were carried out according to the following scheme: (1) control (water foliar spray), (2) Chlorella vulgaris super concentrate suspension ('Organic' ltd, Tolyatti, Samara region, Russia) 35 mL per L, (3) sodium selenate solution, 26.4 mM (50 mg L −1 ), (4) SeCys 2 solution, 26.4 mM (87 mg L −1 ), (5) sodium selenate + Chlorella (the same concentrations), and (6) SeCys 2 + Chlorella (the same concentrations). A split plot design was used for the treatment distribution in the field, with three replicates and each experimental unit covering 10 m 2 . To exclude the interference of other factors, no fertilizers were applied during the experiment. The results were expressed as means of the two-year data.
The plants were sprayed with the appropriate solutions twice: at the stage of head formation (13 July) and 14 days later (4 August).
During the growing season, hoeing and manual weeding were carried out. A 4-fold treatment with insecticides BI-58 New (BASF Societas Europaea, Ludwichshafe, Germany) and Decis-Profi (Bayer Crop Science, Monheim, Germany) was carried out against herbivory.
Cabbage harvesting was carried out on 6-8 October.

Sample Preparation
After harvesting and removal of soil particles from roots, leaves, heads, and roots of at least 10 plants were separated and homogenized. Fresh leaves and heads homogenates were used for the determination of nitrates, ascorbic acid, and photosynthetic pigments. The remaining parts of plants were dried at 70 • C to constant weight and homogenized, and the resulting powders were used for the determination of the total polyphenols content (TP), total antioxidant activity (AOA), and lipids content.

Head Density
Savoy cabbage head density was determined using head mass and head volume, calculated according to [65] using the formula of spheroid volume: where V-head volume, cm 3 ; D-head diameter, cm; H-head height cm.

Dry Matter
The dry matter was assessed gravimetrically by drying the samples in an oven at 70 • C until constant weight.

Ascorbic Acid
The ascorbic acid content was determined by visual titration of leaf and head extracts in 3% trichloracetic acid with sodium 2.6-dichlorophenol indophenolate solution (Tillman's reagent) [67]. Roots were not taken into consideration due to low ascorbic acid content.

Total Polyphenols (TP)
Total polyphenols were determined in 70% ethanol extracts of dried samples using the Folin-Ciocâlteu colorimetric method as previously described [69]. One gram of dry homogenates was extracted with 20 mL of 70% ethanol/water at 80 • C for 1 h. The mixture was cooled down and quantitatively transferred to a volumetric flask, and the volume was adjusted to 25 mL. The mixture was filtered through filter paper, and 1 mL of the resulting solution was transferred to a 25 mL volumetric flask, to which 2.5 mL of saturated Na 2 CO 3 solution and 0.25 mL of diluted (1:1) Folin-Ciocâlteu reagent were added. The volume was brought to 25 mL with distilled water. One hour later the solutions were analyzed through a spectrophotometer (Unico 2804 UV, Suite E Dayton, NJ, USA), and the concentration of polyphenols was calculated according to the absorption of the reaction mixture at 730 nm. As an external standard, 0.02% gallic acid was used. The results were expressed as mg of gallic acid equivalent per g of dry weight (mg GAE g −1 d.w).

Antioxidant Activity (AOA)
The antioxidant activity of cabbage roots, heads, and leaves was assessed on (a) 70% ethanolic extracts of dry samples, and (b) water extracts, using a redox titration method [69]. The values were expressed in mg gallic acid equivalents (mg GAE g −1 d.w.).

Sugars
Monosaccharides were determined using the ferricyanide colorimetric method, based on the reaction of monosaccharides with potassium ferricyanide [70]. Total sugars were analogically determined after acidic hydrolysis of water extracts with 20% hydrochloric acid. Fructose was used as an external standard. The results were expressed in% per dry weight.

Selenium
Selenium was analyzed using the fluorometric method previously described for tissues and biological fluids [71]. Dried homogenized samples were digested via heating with a mixture of nitric-perchloric acids, subsequent reduction of selenate (Se +6 ) to selenite (Se +4 ) with a solution of 6 N HCl, and the formation of a complex between Se +4 and 2,3-diaminonaphtalene. Calculation of the Se concentration was achieved by recording the piazoselenol fluorescence value in hexane at 519 nm λ emission and 376 nm λ excitation. Each determination was performed in triplicate. The precision of the results was verified using a reference standard-lyophilized mitsuba stem in each determination with a Se concentration of 1865 µg·Kg −1 (Federal Scientific Vegetable Center). The results are expressed in µg kg −1 d.w.

Statistical Analysis
Data were processed by analysis of variance, and mean separations were performed through the Duncan's multiple range test, with reference to 0.05 probability level, using SPSS software version 21 (Armonk, NY, USA).

Conclusions
The results of the present research showed that sodium selenate had a greater biofortification effect on Savoy cabbage, compared to SeCys 2 , with the predominance of Se accumulation in leaves, though the growth stimulation effect was higher under organic Se supply which also provided a higher accumulation of photosynthetic pigments. The efficiency of sodium selenate biofortification may be increased with Chlorella foliar supply. Se treatment significantly decreased head density, but improved leaves yield. The beneficial effect of Se on plant yield and leaf AOA reveals the good prospects of Se biofortification in Savoy cabbage.